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Interaction of human intestinal and hepatoma alkaline phosphatases with immobilized cibacron blue F3GA
Vo1.111, No, 1,1983
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 36-40
February 28, 1983
INTERACTION OF HUMAN INTESTINAL AND HEPATOMA ALKALINE PHOSPHATASES WITH IMMOBILIZED CIBACRON BLUE F3GA Hideo Yamamoto, Masanobu Tanaka, Toshikazu Okochi, and Susumu Kishimoto Third Department of Internal Medicine, Osaka University School of Medicine, Fukushima, Osaka 553, Japan Received December 23, 1982
SUMMARY: Possible interactions of human liver and intestinal alkaline phosphatases with Cibacron Blue F3GA were examined. The results indicated that the intestinal enzyme bound to the dye column whereas the liver enzyme did not. The affinity of intestinal alkaline phosphatase with the dye-ligand appeared to be biospecific, since a low concentration of purine nueleoside phosphates or potassium phosphate specifically reversed the binding. Taking advantage of the variant alkaline phosphatase from human hepatocellular cancer tissue to behave on the dye adsorbent in a similar fashion with the intestinal enzyme, it was purified by Cibacron Blue F3GA affinity chromatography, producing a 189fold purification with a yield of 93%.
A sulfonated polyaromatic blue dye, called Cibacron Blue F3GA, complexes with a wide range of proteins with the dinucleotide fold (i).
This structure
is known to form NAD, ATP and other nucleotides binding sites in a variety of enzymes such as lactate dehydrogenase (EC 2.7.2.3).
(EC 1.1.1.27) and phosphoglycerate kinase
The emergence of immobilized Cibacron Blue F3GA as a general
ligand for affinity chromatography has led to new and improved techniques for the purification of enzymes with the dinucleotide fold. Although alkaline phosphatases
(EC 3.1.3.1) from various sources are
designated under one name, they differ in their physical and kinetic character, as well as in their response to activators and inhibitors
(2).
Eaton and Moss
have demonstrated that although the mono-, di- and triphosphates of adenosine or uridine were all active substrates of human liver and small intestinal alkaline phosphatases,
the intestinal enzyme showed greater organic phosphatase
activity relative to its orthophosphatase activity than did the liver enzyme (3). In view of their data, we decided to examine possible interactions of tl~ese two enzymes with Cibaeron Blue FBGA affinity columns.
0006-291X/83/040036-05501.50/0 Copyright © 1983byAcademic Press, ~c. Affrigh~ofreproduet~n ~ anyform rese~ed.
36
The results indicated
Vol. 111, No. 1, 1983
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that the intestinal enzyme specifically under certain conditions whereas of the dye-ligand
bound to and eluted from the dye column
the liver enzyme did not bind.
affinity chromatography
fied in its application
The potency
as a purification method was exempli-
to the variant alkaline phosphatase
Warnock and Reisman in human hepatocellular
cancer
first observed by
(4).
MATERIALS AND METHODS B-NAD, ATP, ADP, AMP, adenosine, GTP, GDP, GMP, guanosine were obtained from Sigma as the sodium salts. L-Phenylalanine and L-tryptophan were from Wako Pure Chemical Industries. Affi-Gel Blue (100-200 mesh) was purchased from Bio-Rad Laboratories. Alkaline phosphatase was assayed in 2 ml of 0.i M carbonate-bicarbonate buffer, pH I0.0, containing i0 mM disodium phenyl phosphate, 3 mM 4-aminoantipyirne, 1 mM MgCI2 and enzyme. The reaction was started by the addition of enzyme. After incubation for 15 min at 37°C, it was terminated by the addition of 2 ml 65 mM acetic acid containing 36 m M potassium ferricyanide. The liberated phenol was determined by measuring the absorbance at 500 nm. One unit was defined as the activity that liberates one umol of phenol per min under the conditions employed. Protein concentration was determined by the method of Lowry et al. (5). Human liver, small intestines and hepatocellular cancer tissue were obtained from autopsy and stored at -70°C until used. The alkaline phosphatases from these sources were partially purified by butanol extraction, acetone precipitation, ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration according to the modified method of Moss et al. (6). The specific activities of the enzyme preparations were 43.1 and 117 units per mg protein for the normal liver and intestinal enzymes, respectively. The variant alkaline phosphatase preparation from DEAE-cellulose chromatography, which had a specific activity of 1.90 units per mg protien, was extensively dialyzed against i0 mM Tris-HCl buffer, pH 7.5, and applied to an Affi-Gel Blue column.
RESULTS The purified human liver alkaline phosphatase was applied to an Affi-Gel Blue column with i0 mM Tris-HCl buffer, approximately appeared
(3 units,
69.6 pg protein)
(1.3 x 7.5 cm), previously equilibrated
pH 7.5, at a flow rate of 2.5 ml per hr.
However,
total activity of the enzyme failed to bind to the column and
in the void volume.
The purified
intestinal
The result was confirmed by using a larger column. alkaline phosphatase
was applied to the same column 40 ml of the equilibration
(3 units, 25.6 ~g protein)
(1.3 × 7.5 cm), and the column was washed with
buffer.
In contrast
to the liver enzyme, nearly
total activity of the intestinal enzyme was retained to the column.
3?
The effect
VOI. 111, No. 1, 1983
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table i.
Effect of eluting agents tested
Elutin$ asent tested
Concentration
NAD ATP, ADP, AMP GTP, GDP, GMP adenosine, guanosine potassium phosphate L-phenylalanine, L-tryptophan Tris-IICI, pH 7.5 Tris-HCI, pH 7.5
(mM)
Elution
i 1 i i 1 5 50 i00
No Yes Yes No Yes No No Yes
A series of Affi-Gel Blue columns (1.3 X 7.5 cm) were equilibrated with i0 mM Tris-HCl buffer, pH 7.5. The intestinal alkaline phosphatase preparation (3 units, 25.6 Dg protein) was applied to each of the columns at a flow rate of 2.5 ml per hr. After the column was washed with 40 ml of the equilibration buffer, the enzyme was eluted by the addition of indicated concentrations of each eluting agent to the same buffer. A total of 40 ml eluate was collected in 2-ml fractions.
of the tested
eluting
or triphosphates phosphate readily
agents
of adenosine
in i0 m M Tris-HCI,
eluted
each case.
in a single
A pulse
representing
phosphates
stereospecific
were not effective
' Cibacron
of the variant
tissue.
The enzyme
column
(1.6 X i0 cm),
and recycled
to assure
level
as a ligand
equilibrated
adenosine
nor
L-Phenylalanine alkaline
phos-
for the c h r o m a t o g r a p h i c
puri-
intestinal
from human h e p a t o c e l l u l a r was applied with
cancer
to an A f f i - G e l
i0 m M Tris-HCI
of the enzyme
with 60 ml of the e q u i l i b r a t i o n
phosphate
containing
buffer,
Blue pH
for 24 hr at a flow rate of I0 ml per hr
of binding
off the column,
in
very gradually,
Neither
the enzyme.
100%
either.
phosphatase
the column
the enzyme
phosphate.
of human
20 mg protein)
previously
through
peak eluted
1 mM p o t a s s i u m
alkaline
agents
the enzyme
than i0 m M Tris-HCl
to eluate
inhibitors
eluting
(38 units,
an increased
column was washed protein
or p o t a s s i u m
agent,
di-
of potassium
was approximately
pH 7.5 eluted
less effective
Blue F3GA was examined
fication
7.5,
was
When i m M mono-,
as an eluting
The recovery
nor NAD at 1 m M was not able
and L-tryptophan, phatase,
peak.
i.
or the same c o n c e n t r a t i o n
pH 7.5 was used
sharp
i00 mM Tris-HCl
in Table
or guanosine,
of I00 m M Tris-HCI,
1 r~i purine nucleoside guanosine
is summarized
the enzyme was
to the same buffer
38
activity
buffer.
applied.
After
the initial
eluted by the a d d i t i o n
(Fig.l).
The
The
specific
of
activity
of
Vol. 111, No. 1, 1983
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNI£ATIONS
20
I mM potassium ~ phosphate
IJJ
O3 < "r'o n r-
1.0
0 "r~ I0 13_...
._I ,<~
',
5
o.s
i
J
"--"-" . . . . . . . . . .
":
--
""
I0 20 FRACTION NUMBER
0
0
30
Fig. I. Elution profile of variant alkaline phosphatase from Affi-Gel Blue. An Affi-Gel Blue column (1.6× l0 cm) was equilibrated with i0 mM TrisHCI buffer, pIl 7.5. The enzyme preparation (38 units, 20 mg protein) was applied to the column, and recycled through the column for 24 hr at a flow rate of i0 ml per hr. The column was then washed with 60 ml of the equilibration buffer, and the enzyme was eluted with i mM potassium phosphate in the same buffer at a flow rate of 4 ml per hr. 4-ml fractions were collected. Dotted line, absorbance at 280 nm; solid circles, enzyme in units/fraction.
the enzyme 189-fold
eluted
from the column was
purification
with a yield
359 units
per mg protein,
representing
a
of 93%.
DISCUSSION We have
studied
three human
hepatocellular
cancer
tissue,
hepatoblastoma
tissue
(7).
immunologically degree, tical,
suggesting although
tibility finding bound
identical that
and HUH-6,
These
three
and inhibited
to neuraminidase
phosphatases a cultured
alkaline
in m o l e c u l a r
isoenzymes
phosphatases
weight,
Blue F3GA affinity
amino
The most
column
proved acids
interesting
type alkaline
whereas
liver
from
to be
to an equal
sites must be iden-
thermostability
of intestinal
intestine,
cell line e s t a b l i s h e d
of their catalylic
(data not shown).
that all of these
from small
by the various
the a r c h i t e c t u r e
they differed
to a Cibacron
alkaline
and suscepresult
is the
phosphatase
alkaline
phosphatase
did not. The precise phosphatases appear
mechanism
for the i n t e r a c t i o n s
w i t h the dye cannot
to be biospecific
be fully
in nature
and not
39
of the intestinal
explained. ionic,
However,
since
type alkaline
these interactions
the ionic
strength
of
Vol. 111, No. 1, 1983
Tris-NCl required
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to eluate the enzymes was much higher than the ionic strength
of purine nucleoside phosphates or potassium phosphate required for elution. In addition,
ionic interactions between these acidic glycoproteins
anionic dye are unlikely to occur. interactions
Furthermore,
that the dye-enzyme complexes
of salt, and broken in high concentrations
and the
it indicates non-hydrophobic
could be formed in low concentrations of salt, since hydrophobic
inter-
actions are generally more intense at higher salt concentrations. Bouriotis
and Dean have examined Cibacron Blue 3GA-Sepharose
graphy as a method for the purification tine (8). extract,
of alkaline phosphatase
6B chromato-
from calf intes-
They obtained a 17-, 128- or 290-fold purification over the initial using gradients of potassium chloride or pulsed elution with i0 mM
inorganic phosphate or the same concentration enzyme protein, chromatography
respectively.
of ~-naphthyl
They have concluded
phosphate to desorb
that the dye-ligand
affinity
coupled with the use of affinity elution would provide a useful
purification method for alkaline phosphatase
from calf intestine extracts.
Our
present preliminary data have also shown that Cibacron Blue F3GA affinity chromatography will be a useful method for the purification human intestinal type alkaline phosphatase. phosphatases
of various isoenzymes
The variant and liver alkaline
often coexist in sera or cancer tissues of patients with hepato-
cellular cancer.
This dye-ligand affinity chromatography will be particularly
useful in separating
the former enzyme from the latter.
REFERENCES i. 2. 3. 4. 5. 6. 7. 8.
Sundaram, P.V., and Eckstein, F. (1978) Theory and Practice in Affinity Techniques, pp. 23-44, Academic Press, New York. Fishman, W.H. (1974) Am. J. Med. 56, 617-650. Eaton, R.H., and Moss, D.W. (1967) Biochem. J. 105, 1307-1312. Warnock, M.L., and Reisman, R. (1969) Clin. Chim. Acta. 24, 5-11. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Moss, D.W., Eaton, R.H., Smith, J.K., and Whitby, L.G. (1967) Biochem. J. 102, 53-57. Doi, I. (1976) Gann. 67, i-i0. Bouriotis, V., and Dean, P.D.G. (1981) J. Chromatogr. 206, 521-530.
40
of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 36-40
February 28, 1983
INTERACTION OF HUMAN INTESTINAL AND HEPATOMA ALKALINE PHOSPHATASES WITH IMMOBILIZED CIBACRON BLUE F3GA Hideo Yamamoto, Masanobu Tanaka, Toshikazu Okochi, and Susumu Kishimoto Third Department of Internal Medicine, Osaka University School of Medicine, Fukushima, Osaka 553, Japan Received December 23, 1982
SUMMARY: Possible interactions of human liver and intestinal alkaline phosphatases with Cibacron Blue F3GA were examined. The results indicated that the intestinal enzyme bound to the dye column whereas the liver enzyme did not. The affinity of intestinal alkaline phosphatase with the dye-ligand appeared to be biospecific, since a low concentration of purine nueleoside phosphates or potassium phosphate specifically reversed the binding. Taking advantage of the variant alkaline phosphatase from human hepatocellular cancer tissue to behave on the dye adsorbent in a similar fashion with the intestinal enzyme, it was purified by Cibacron Blue F3GA affinity chromatography, producing a 189fold purification with a yield of 93%.
A sulfonated polyaromatic blue dye, called Cibacron Blue F3GA, complexes with a wide range of proteins with the dinucleotide fold (i).
This structure
is known to form NAD, ATP and other nucleotides binding sites in a variety of enzymes such as lactate dehydrogenase (EC 2.7.2.3).
(EC 1.1.1.27) and phosphoglycerate kinase
The emergence of immobilized Cibacron Blue F3GA as a general
ligand for affinity chromatography has led to new and improved techniques for the purification of enzymes with the dinucleotide fold. Although alkaline phosphatases
(EC 3.1.3.1) from various sources are
designated under one name, they differ in their physical and kinetic character, as well as in their response to activators and inhibitors
(2).
Eaton and Moss
have demonstrated that although the mono-, di- and triphosphates of adenosine or uridine were all active substrates of human liver and small intestinal alkaline phosphatases,
the intestinal enzyme showed greater organic phosphatase
activity relative to its orthophosphatase activity than did the liver enzyme (3). In view of their data, we decided to examine possible interactions of tl~ese two enzymes with Cibaeron Blue FBGA affinity columns.
0006-291X/83/040036-05501.50/0 Copyright © 1983byAcademic Press, ~c. Affrigh~ofreproduet~n ~ anyform rese~ed.
36
The results indicated
Vol. 111, No. 1, 1983
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that the intestinal enzyme specifically under certain conditions whereas of the dye-ligand
bound to and eluted from the dye column
the liver enzyme did not bind.
affinity chromatography
fied in its application
The potency
as a purification method was exempli-
to the variant alkaline phosphatase
Warnock and Reisman in human hepatocellular
cancer
first observed by
(4).
MATERIALS AND METHODS B-NAD, ATP, ADP, AMP, adenosine, GTP, GDP, GMP, guanosine were obtained from Sigma as the sodium salts. L-Phenylalanine and L-tryptophan were from Wako Pure Chemical Industries. Affi-Gel Blue (100-200 mesh) was purchased from Bio-Rad Laboratories. Alkaline phosphatase was assayed in 2 ml of 0.i M carbonate-bicarbonate buffer, pH I0.0, containing i0 mM disodium phenyl phosphate, 3 mM 4-aminoantipyirne, 1 mM MgCI2 and enzyme. The reaction was started by the addition of enzyme. After incubation for 15 min at 37°C, it was terminated by the addition of 2 ml 65 mM acetic acid containing 36 m M potassium ferricyanide. The liberated phenol was determined by measuring the absorbance at 500 nm. One unit was defined as the activity that liberates one umol of phenol per min under the conditions employed. Protein concentration was determined by the method of Lowry et al. (5). Human liver, small intestines and hepatocellular cancer tissue were obtained from autopsy and stored at -70°C until used. The alkaline phosphatases from these sources were partially purified by butanol extraction, acetone precipitation, ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration according to the modified method of Moss et al. (6). The specific activities of the enzyme preparations were 43.1 and 117 units per mg protein for the normal liver and intestinal enzymes, respectively. The variant alkaline phosphatase preparation from DEAE-cellulose chromatography, which had a specific activity of 1.90 units per mg protien, was extensively dialyzed against i0 mM Tris-HCl buffer, pH 7.5, and applied to an Affi-Gel Blue column.
RESULTS The purified human liver alkaline phosphatase was applied to an Affi-Gel Blue column with i0 mM Tris-HCl buffer, approximately appeared
(3 units,
69.6 pg protein)
(1.3 x 7.5 cm), previously equilibrated
pH 7.5, at a flow rate of 2.5 ml per hr.
However,
total activity of the enzyme failed to bind to the column and
in the void volume.
The purified
intestinal
The result was confirmed by using a larger column. alkaline phosphatase
was applied to the same column 40 ml of the equilibration
(3 units, 25.6 ~g protein)
(1.3 × 7.5 cm), and the column was washed with
buffer.
In contrast
to the liver enzyme, nearly
total activity of the intestinal enzyme was retained to the column.
3?
The effect
VOI. 111, No. 1, 1983
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table i.
Effect of eluting agents tested
Elutin$ asent tested
Concentration
NAD ATP, ADP, AMP GTP, GDP, GMP adenosine, guanosine potassium phosphate L-phenylalanine, L-tryptophan Tris-IICI, pH 7.5 Tris-HCI, pH 7.5
(mM)
Elution
i 1 i i 1 5 50 i00
No Yes Yes No Yes No No Yes
A series of Affi-Gel Blue columns (1.3 X 7.5 cm) were equilibrated with i0 mM Tris-HCl buffer, pH 7.5. The intestinal alkaline phosphatase preparation (3 units, 25.6 Dg protein) was applied to each of the columns at a flow rate of 2.5 ml per hr. After the column was washed with 40 ml of the equilibration buffer, the enzyme was eluted by the addition of indicated concentrations of each eluting agent to the same buffer. A total of 40 ml eluate was collected in 2-ml fractions.
of the tested
eluting
or triphosphates phosphate readily
agents
of adenosine
in i0 m M Tris-HCI,
eluted
each case.
in a single
A pulse
representing
phosphates
stereospecific
were not effective
' Cibacron
of the variant
tissue.
The enzyme
column
(1.6 X i0 cm),
and recycled
to assure
level
as a ligand
equilibrated
adenosine
nor
L-Phenylalanine alkaline
phos-
for the c h r o m a t o g r a p h i c
puri-
intestinal
from human h e p a t o c e l l u l a r was applied with
cancer
to an A f f i - G e l
i0 m M Tris-HCI
of the enzyme
with 60 ml of the e q u i l i b r a t i o n
phosphate
containing
buffer,
Blue pH
for 24 hr at a flow rate of I0 ml per hr
of binding
off the column,
in
very gradually,
Neither
the enzyme.
100%
either.
phosphatase
the column
the enzyme
phosphate.
of human
20 mg protein)
previously
through
peak eluted
1 mM p o t a s s i u m
alkaline
agents
the enzyme
than i0 m M Tris-HCl
to eluate
inhibitors
eluting
(38 units,
an increased
column was washed protein
or p o t a s s i u m
agent,
di-
of potassium
was approximately
pH 7.5 eluted
less effective
Blue F3GA was examined
fication
7.5,
was
When i m M mono-,
as an eluting
The recovery
nor NAD at 1 m M was not able
and L-tryptophan, phatase,
peak.
i.
or the same c o n c e n t r a t i o n
pH 7.5 was used
sharp
i00 mM Tris-HCl
in Table
or guanosine,
of I00 m M Tris-HCI,
1 r~i purine nucleoside guanosine
is summarized
the enzyme was
to the same buffer
38
activity
buffer.
applied.
After
the initial
eluted by the a d d i t i o n
(Fig.l).
The
The
specific
of
activity
of
Vol. 111, No. 1, 1983
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNI£ATIONS
20
I mM potassium ~ phosphate
IJJ
O3 < "r'o n r-
1.0
0 "r~ I0 13_...
._I ,<~
',
5
o.s
i
J
"--"-" . . . . . . . . . .
":
--
""
I0 20 FRACTION NUMBER
0
0
30
Fig. I. Elution profile of variant alkaline phosphatase from Affi-Gel Blue. An Affi-Gel Blue column (1.6× l0 cm) was equilibrated with i0 mM TrisHCI buffer, pIl 7.5. The enzyme preparation (38 units, 20 mg protein) was applied to the column, and recycled through the column for 24 hr at a flow rate of i0 ml per hr. The column was then washed with 60 ml of the equilibration buffer, and the enzyme was eluted with i mM potassium phosphate in the same buffer at a flow rate of 4 ml per hr. 4-ml fractions were collected. Dotted line, absorbance at 280 nm; solid circles, enzyme in units/fraction.
the enzyme 189-fold
eluted
from the column was
purification
with a yield
359 units
per mg protein,
representing
a
of 93%.
DISCUSSION We have
studied
three human
hepatocellular
cancer
tissue,
hepatoblastoma
tissue
(7).
immunologically degree, tical,
suggesting although
tibility finding bound
identical that
and HUH-6,
These
three
and inhibited
to neuraminidase
phosphatases a cultured
alkaline
in m o l e c u l a r
isoenzymes
phosphatases
weight,
Blue F3GA affinity
amino
The most
column
proved acids
interesting
type alkaline
whereas
liver
from
to be
to an equal
sites must be iden-
thermostability
of intestinal
intestine,
cell line e s t a b l i s h e d
of their catalylic
(data not shown).
that all of these
from small
by the various
the a r c h i t e c t u r e
they differed
to a Cibacron
alkaline
and suscepresult
is the
phosphatase
alkaline
phosphatase
did not. The precise phosphatases appear
mechanism
for the i n t e r a c t i o n s
w i t h the dye cannot
to be biospecific
be fully
in nature
and not
39
of the intestinal
explained. ionic,
However,
since
type alkaline
these interactions
the ionic
strength
of
Vol. 111, No. 1, 1983
Tris-NCl required
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to eluate the enzymes was much higher than the ionic strength
of purine nucleoside phosphates or potassium phosphate required for elution. In addition,
ionic interactions between these acidic glycoproteins
anionic dye are unlikely to occur. interactions
Furthermore,
that the dye-enzyme complexes
of salt, and broken in high concentrations
and the
it indicates non-hydrophobic
could be formed in low concentrations of salt, since hydrophobic
inter-
actions are generally more intense at higher salt concentrations. Bouriotis
and Dean have examined Cibacron Blue 3GA-Sepharose
graphy as a method for the purification tine (8). extract,
of alkaline phosphatase
6B chromato-
from calf intes-
They obtained a 17-, 128- or 290-fold purification over the initial using gradients of potassium chloride or pulsed elution with i0 mM
inorganic phosphate or the same concentration enzyme protein, chromatography
respectively.
of ~-naphthyl
They have concluded
phosphate to desorb
that the dye-ligand
affinity
coupled with the use of affinity elution would provide a useful
purification method for alkaline phosphatase
from calf intestine extracts.
Our
present preliminary data have also shown that Cibacron Blue F3GA affinity chromatography will be a useful method for the purification human intestinal type alkaline phosphatase. phosphatases
of various isoenzymes
The variant and liver alkaline
often coexist in sera or cancer tissues of patients with hepato-
cellular cancer.
This dye-ligand affinity chromatography will be particularly
useful in separating
the former enzyme from the latter.
REFERENCES i. 2. 3. 4. 5. 6. 7. 8.
Sundaram, P.V., and Eckstein, F. (1978) Theory and Practice in Affinity Techniques, pp. 23-44, Academic Press, New York. Fishman, W.H. (1974) Am. J. Med. 56, 617-650. Eaton, R.H., and Moss, D.W. (1967) Biochem. J. 105, 1307-1312. Warnock, M.L., and Reisman, R. (1969) Clin. Chim. Acta. 24, 5-11. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Moss, D.W., Eaton, R.H., Smith, J.K., and Whitby, L.G. (1967) Biochem. J. 102, 53-57. Doi, I. (1976) Gann. 67, i-i0. Bouriotis, V., and Dean, P.D.G. (1981) J. Chromatogr. 206, 521-530.
40
of